Previous research from our laboratory has revealed dramatic differences in the targeting of several apical and basolateral plasma membrane proteins between Retinal Pigment Epithelium (RPE) and other simple epithelia. The model apical protein, Influenza Hemagglutinin (HA), follows a transcytotic route in RPE rather than the conventional direct route between Trans Golgi Network (TGN) and apical cell surface observed in other epithelial cells. Basolateral proteins such as NCAM-140 and RET-PE2 (EMMPRIN) have a strikingly reversed apical polarity in adult RPE but the latter is basolateral in the immature RPE of newborn rats. This proposal addresses the fundamental mechanisms involved in the establishment of polarity in RPE. Experiments in aim l will characterize the role of components of the apical sorting machinery, such as caveolin, MAL proteolipid, lectin and non-lectin receptors in the targeting of HA and other apical proteins in RPE (aim 1). Experiments in aim 2 will explore the mechanisms responsible for the apical reversal of NCAM and EMMPRIN. To accomplish this, we will analyze the role of known adaptor complexes (AP1, AP3, AP4), of ubiquilin (a protein that associates with the basolateral signal of NCAM) and of recently identified TGN-associated adaptors, in the basolateral sorting of these molecules. We will identify the basolateral signal of EMMPRIN and, as this protein is ubiquitinated, determine the role of ubiquitination in basolateral targeting. Experiments in aim 3 will continue to characterize the role of ezrin in RPE morphogenesis. We have recently shown that ezrin is key to the development of the very long apical microvilli and the highly convoluted basal infoldings during maturation of RPE in newborn rats. We will search for binding partners of ezrin at apical and basolateral surfaces and assess their specific roles in these processes. We will determine how ezrin is activated by phosphorylation and determine the role of kinases and small GTPases that participate in this process. We will study how blocking ezrin synthesis in vivo affects the maturation of RPE microvilli and associated photoreceptors. The information obtained should help understand the role of RPE in a variety of blinding diseases, such as age-related macular degeneration and diabetic retinopathy.